A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

Summary

A gentle touch-force loading machine is built from human hair brushes, robotic arms and a controller. The hair brushes are driven by robotic arms installed on the machine and move periodically to apply touch-force on plants. The strength of machine-driven hair touches is comparable to that of manually applied touches.

Abstract

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called thigmomorphogenesis. In past decades, several signaling components have been identified and reported for being involved in the mechanotransduction (e.g., calcium ion binding proteins and jasmonic acid biosynthesis enzymes). However, the relatively slow pace of research in the study of force signaling or thigmomorphogenesis is largely attributed to two reasons: the requirement for laborious human hand-manipulated touch induction of thigmomorphogenesis and the force strength errors associated with people’s hand-touch. To enhance the efficiency of external force loading on a plant organism, an automatic touch-force loading machine was built. This robotic arm-driven hair brush touches provide a labor-saving and easily repeatable touch-force simulation, unlimited rounds of touch repetition and adjustable touch strength. This hair touch-force loading machine can be used for both large scale screening of touch-force signaling mutants and the phenomics study of plant thigmomorphogenesis. In addition, touch materials such as human hair, can be replaced with other natural materials like animal hair, silk threads and cotton fibers. The automated moving arms on the machine may be equipped with water sprinkling nozzles and air blowers to mimic the natural forces of rain drops and wind, respectively. By using this automatic hair touch-force loading machine in combination with the hand-performed cotton swab touching, we have investigated the touch response of two force signaling mutants, MAP KINASE KINASE 1 (MKK1) and MKK2 plants. The phenomes of the touch-force loaded wild type plants and two mutants were evaluated statistically. They have exhibited significant differences in touch response.

Introduction

Plant thigmomorphogenesis is a term that was coined by Jaffe, MJ in 1973¹. It is a plant tropism but different from the well-known phototropism or gravitropism caused by stimuli of sunlight or gravity^2,3. It describes phenotypic alterations associated with periodic mechanical stimulations, which have been frequently observed by botanists in earlier times^4,5. Raindrops, wind, plant, animal and human touches, even animal bites, are all considered to be different types of mechano-stimuli that trigger the force signaling in plants^4,5. Characteristics of plant thigmomorphogenesis include the delay of bolting, a shorter stem, smaller rosette/leaf size in herbaceous plants, and thicker stem in woody plants^6,7,8. This is unlike the thigmonastic or thigmotropic response often found in the Mimosa plant or other mechano-sensitive vines, where these rapid touch responses are easier to be observed^1,9,10. Thigmomorphogenesis, on the other hand, is relatively difficult to be observed because of its slow growth response. Thigmomorphogenesis is usually observed following weeks or even years of continuous force-loading stimulation. This unique nature of plant touch response makes it difficult to perform a forward genetic screen using human hand touch stimulation to isolate the touch-force signaling resistant mutants in a robust manner.

To elucidate the force signal transduction pathways and the molecular mechanisms underlying the thigmomorphogenesis^6,11, molecular and cellular biological experiments have been performed in the past^6,12,13,14. These studies have proposed that the plant force signal receptors mainly consist of mechanosensitive ion channels (MSC) and the tethered MSC complexes composed by multimeric complexes of membrane-spanning proteins^11,14,15. The cytoplasmic Ca²⁺ transient spike generated within seconds of the initial touch. Wind-, rain-, or gravi-stimulation may interact with the downstream calcium sensors to transduce the force signals to nuclear events^14,16,17,18. In addition to molecular and cellular studies, the forward genetic screen with manual finger touching of plants has found that phytohormones and the secondary metabolites are involved in the consequent touch-inducible (TCH) gene expression following the touch-force loading^13,19. For examples, aos and opr3²⁰ mutants have been identified thus far from the genetic studies. However, the major problem associated with application of the forward genetics in the study of thigmomorphogenesis is still the intensive labor required for quantitating the level of touch response and touching a large population of genetically mutated individual plants. The time-consuming issue also persists in the hand touching-based mutant screen^14,20. For an example, to complete one round of touch-force stimulation, a person needs to touch 30-60 times (one touch per second) on an individual plant. In order to have enough number of plants for statistical phenotype analysis, 20-50 individual plants of the same genotype are normally required for the touch-force loading process. This touch-force loading regime means that a person needs to repetitively perform 600-3,000 touches on one genotype of choice. This type of touch normally needs to be repeated 3 to 5 rounds a day, which equals roughly 1,800-15,000 finger or cotton swab touches per day per genotype of plants. A well-trained person is normally required to maintain the strength and force of multiple touches within a desirable range throughout many rounds of repetition in a day to avoid the large variation in force and strength. As it is well known that thigmomorphogenesis is a saturable and dose-dependent process^6,21, touch force/strength becomes critical to a success in triggering touch response of a plant.

To remove the person-dependent touch-force loading and to maintain mechanical application within an acceptable error range¹⁴, we therefore designed an automatic touch-force loading machine to replace the hand-manipulated touches. The machine has 4 moving arms built, each of which is equipped with one human hair brush. This version is named Model K1 to specify its feature of human hair touch-force loading. If 4 genotypes are measured quantitatively for their thigmomorphogenesis or touch response under one machine, 40-48 individuals per genotype can be measured. Each round of touch repetition (less than 60 times of touch per plant) lasts less than 5 minutes using a moving speed adjustable robotic arm. Thus, plants on a Model K1 touch machine can be mechanically stimulated for multiple rounds a day either with a constant touch-force loading or different levels of strengths as initially programmed.

Arabidopsis thaliana, a model plant organism, was therefore chosen as the target plant species for testing the fully automatic hair touch-force loading machine application. Because there are several large seedbanks available for retrieving the various germplasms of mutants and the size of flowering, Arabidopsis fits well to the space available in the growth shelf mounted with the Model K1 touch machine.

The Model K1 automatic touch machine consists of three major components: (1) the H-shape metal rack composed by two belt-driven linear actuators, (2) robotic metal arms equipped with hair brushes, and (3) a controller. For a customized Model K1 touch machine, each X/Y axis module is composed of one belt-driven guide-rail, two slide blocks (red) and one 57 stepper motor (pre-installed and dismountable) (Figure 1A,B). The upper horizontal actuator allows the robotic metal arm to move left and right horizontally, the lower vertical belt-driven linear actuator allows the robotic metal arm to move up and down vertically (Figure 1B, Figure 2A). Four dismountable robotic arms were installed on the vertical actuator (Figure 1C, Figure 2B). Four human hair brushes were bound to four robotic arms, respectively (Figure 1C, Figure 2B). All mechanical parts to construct the Model K1 touch machine in bolded font below are marked in Figure 1C (also see the Table of Materials).

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Protocol

1. Seed preparation

NOTE: Arabidopsis seeds of both wild type (Col-0) as well as mkk1 and mkk2 loss-of-function mutants used were purchased from the Arabidopsis Biological Resource Center (ABRC, https://www.arabidopsis.org, Columbus, OH).

1. Calculate how many plant individuals of each genotype will be used for a reliable statistical analysis. Prepare a sufficient number of seeds based on the germination rate of each line, usually 4-5 times more than what is needed for an experiment. Ensure enough number of healthy and uniform-sized plants can be used for touch response assay. According to this protocol, 300-500 seeds per genotype are usually used to produce 80-90 plants of similar size.
2. Immerse seeds in cold water and store them in 4 °C (covered with aluminum foil to keep in dark) for seed imbibition. Sow the seeds 5-7 days after the imbibition.

2. Plant growth

1. Select the appropriate soil for plant growth (see the Table of Materials). Avoid large clumps and mix them homogenously.
2. Prepare 24 plastic cups: the holding capacity is 207 mL and the upper rim diameter is 7.4 cm. Drill three round holes at the bottom of a cup for irrigation purpose.
3. Fill these plastic cups with the mixed soil. Let the soil piling up to 1-2 cm higher than the cup rim and flatten the surface of piled soil softly.
4. Transfer 24 cups into a plastic tray (21 inches x 10.8 inches x 2.5 inches) and place the tray under constant light condition (see below).
5. Add 2.5 L of water into each tray two hours before seed sowing. Let the soil to absorb the water from holes located at the bottoms of cups and wait for the surface of the soil to drop to the cup rim level.
6. Sow 3-4 seeds into a single spot, and 4 evenly distributed spots within a cup.
7. Place a transparent plastic cover above each tray, and let seeds germinate for a week. Then remove the cover and allow seedlings to grow for another week.
8. Remove extra plants by thinning and keep 4 plant individuals of similar size in each cup 9-10 days after seed sowing.
9. Irrigate plants with 1.5 L of water every other day after the seeds germinate.

3. Growth condition

1. Set the temperature of the growth chamber at 23.5 ± 1.5 °C, and humidity between 35 and 45%.
2. Set the light intensity between 180 and 240 μE∙m^-2∙s^-1 (measured by IL 1700 research radiometer, International Light)¹⁴. The photosynthetic active radiation is from 90 to 120 μE∙m^-2∙s^-1.
3. Set the light condition to be 24 h constant.

4. The construction of touch-force loading machine

NOTE: This robotic hair touch-force loading machine (Model K1) is designed to serve purposes of both touch-force signaling mutant screening and plant thigmomorphogenesis generation (Figure 1, Figure 2).

1. Pre-installation modules (dismountable, Figure 1C)
   1. Install two slide blocks (I) and one 57 stepper motor (II) onto the X/Y axis guide-rail module (III/V).
   2. Install two slide blocks (I) onto the X/Y axis auxiliary girder (IV/VI).
2. Installation of other mechanical parts (Figure 1C)
   1. Fix the X axis guide-rail module (III) and X axis auxiliary girder (IV) together by assembling two junction plates (VII) at each end of the guide-rail.
   2. Fix the Y axis guide-rail module (V) onto the dorsal of two slide blocks (X axis) in a crossing position by assembling two junction plates (VIII) in between.
   3. Fix the Y axis auxiliary girder (VI) onto the dorsal of the other two slide blocks (X axis) in a crossing position by assembling two junction plates (VIII) in between.
   4. Assemble the holder of robot arms (IX) onto the front of two slide blocks (Y axis) in a crossing position with a junction plate (Figure 2A).
   5. Assemble 4 hair brushes (X) onto robot arms (IX) with clamps (Figure 2B).

5. Touch-Force loading machine setting

NOTE: All of controlling parameters to set the Model K1 touch machine in bolded font below are shown in the control panel (Figure 2F).

1. Install touch hair brushes onto the robotic arms. Use a 330 mm-long steel ruler as a holder to fix one layer of human hair (3,600-4,600 hairs/brush) evenly. The length of the hair is 126 mm (Figure 1C).
2. Fix those steel rulers onto the robotic arms with two metal clamps.
3. Set the height of machine arms along the vertical dimension (Y axis) first. Press Jog F+ to raise and Jog R- to lower the robotic arms and brushes. Let the tip of hair brushes 0.5 cm lower than the cup rim. Press the ZERO set. Pre-run the machine 1-2 cycles to make sure all plant individuals are being touched. Adjust and calibrate the brushes and hair tips to the same height every day during the entire touching period.
4. Use an electronic scale to measure the touch force (vertical loading) and maintain the touch force level at 1-2 mN¹⁴.
5. Set the starting position of machine arms along the horizontal dimension (X axis) manually. Allow the hair brushes to hang at the edge of each tray and make sure that no plant is being touched before the touching experiment starts. Press Jog F+/Jog R- to move the machine arm horizontally little by little to set the starting position.
6. Set the hair brush traveling distance in the horizontal dimension (X axis) to 365 mm by pressing the Travel button. Press Inc. F+/Inc. R- to move the machine arms to obtain a full travel distance and ensure that all of the treated plants are being touched during the entire touching experiment.
7. Set the movement speed along the X axis of the machine arms at 5,000 mm/min by pressing the Auto Speed button. Keep the same movement speed during the entire touching experiment.
8. Set the touch time at 20 trials by pressing the Minor Cycle button. Keep the same number of touches per round during the entire touching experiment.
   NOTE: One Minor Cycle equals two Travel distances, which means machine arms will move from the starting position to the end position and then back to the starting position. One minor cycle generates two touches. Hair brushes touch plants 40 times within 20 trials (2 touches x 20 trials = 40 touches). The 40-touch is defined to be one round of touch-force loading.
9. Set the repetition interval of the touch-round at 480 min per day by pressing the Major Period button. Keep the same frequency of touch rounds during an entire touching experiment.
   NOTE: This allows hair brushes to touch plants for 3 rounds a day, and the interval time between each round is 480 min (8 h). The displayed blue number stands for the interval time of each touch round. The machine will start a new round of touch automatically when the countdown below (red number) turns to 0000.
10. Set the Major Cycle at 12 trials, which means that the machine will touch plants for 12 rounds within a period of 4 days automatically. This setting of 12 trials is used to avoid human error in skipping a day of touching.
11. Press the start button to initiate the pre-set program. The Model K1 touch machine will automatically perform the touch force loading according to settings.

6. Physiological data collection and analysis

1. Days to Bolting: Record the bolting day of each plant individually within a touching experiment. Bolting is a symbol that a plant changes its growth stage from the vegetative phase to the reproductive phase. In Arabidopsis, the bolting day is defined as the number of days used by a plant to have its first inflorescence stem reach 1 cm in length.
   NOTE: Under the growth condition described above, the bolting of wild type plants normally initiates from 19 to 23 days after seed sowing and ends at 28-32 days.
2. Rosette Radius: Measure the distance from the rosette center to the tip of the longest leaf.
   1. Take photos of the whole tray from the top. Take photos of the control group and the touch-treated group separately.
   2. Download the appropriate software. Use the free downloaded software ImageJ (https://imagej.nih.gov/ij/download.html) for example.
   3. Open a photo file, use the zoom function to zoom the photo into an appropriate size.
   4. Choose the Straight tool to draw a straight line between the rosette center and tip of a longest leaf to measure the rosette radius.
   5. Select one plant and press the left button to draw a straight line from the rosette center to the longest leaf tip.
   6. Choose the Analyze-Measure function or press Ctrl + M to analyze the line distance.
   7. Select one cup and repeat the previous two steps to analyze the diameter of each plastic cup at the same time. Use these data to perform the calculation to eliminate the bias resulting from the photo-taking.
      NOTE: The equation is:
      R_a/D_a = R_m/D_m
      (R_a, the actual Rosette Radius of a plant; D_a, the actual diameter of plastic cup; R_m, the measured Rosette Radius of the same plant determined by a software; D_m, the measured diameter of the plastic cup which is used for growing the same plant)
3. Rosette Area: Measure the horizontal 2-dimensional surface area of rosette leaves.
   1. Remove the inflorescence without affecting the rest of rosette organs.
   2. Take photos from the top of each plant together with a scale ruler placed nearby.
   3. Use one free plugin of ImageJ, Rosette Tracker and follow the protocol published previously²².

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Results

The automatic hair touch-force loading machine
For observation of morphological changes on plants, both the reproducible growth conditions and treatment methods are key to obtaining repeatable results. This high-throughput and automatic touch-force signaling mutant screening is achieved by the newly built hair touch-force loading machine, Model K1 (Figure 1, Figure 2). These hair brushes can touch a maximum of 4 trays of plants simultaneou...

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Discussion

Thigmomorphogenesis is a complex plant growth response towards mechanical perturbations, which involves a network of cellular signaling and action of phytohormones. It is a consequence of adaptive evolution of plants to survive under the undesirable environmental conditions^25,26. Mechanical touch, especially human finger touch and hand-held cotton swab touch, have been selected to study this morphological changes in previously thigmomorphogenetic studies

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Materials

Name	Company	Catalog Number	Comments
4 hair brushes		customized
4 robot arms with one holder		customized	1000 mm length holder and 560 mm length robot arm
57 stepper motor		57HS22-A
All purpose potting soil	Plantmate, Hong Kong
Arabidopsis plant seeds	Arabidopsis Biological Resource Centers, Columbus, OH		For arabidopsis seed purchase
BIO-MIX potting substratum	Jiffy Products International BV, the Netherlands	1000682050	Two soils were mixed together to grow Arabidopsis. The ratio of All purpos potting soil and  BIO-MIX is 1:2
IL 1700 research radiometer	International Light, Newburyport, MA		The light intensity of both full-wavelength and photosynthetic active radiation can be measured.
ImageJ	https://imagej.nih.gov/ij/download.html		Free downloaded software
Ju Feng Precision and Automation Technology Limited	Shenzhen, China		For belt-driven linear actuators and other mechanical modules purchase
Junction plate of the slide block			To fix the Y guide-rail module or Y auxiliary girder onto backs of slide blocks
Junction plate of the X axis module		customized	To connect the X guide-rail module and X auxiliary girder
Slide block
WDT4045 X axis guide-rail module		843 mm, customized	Pre-installed with two slide blocks and one 57 stepper motor
WDT4045 Y axis guide-rail module		1038 mm, customized	Pre-installed with two slide blocks and one 57 stepper motor
X axis auxiliary girder		843 mm, customized	Pre-installed with two slide blocks
Y axis auxiliary girder		1038 mm, customized	Pre-installed with two slide blocks